Method for extracting nucleic acids from a wide range of organisms
Methods for extracting nucleic acids by heating sample cells at about 80-95 degrees C. in a permeabilization reagent containing a non-ionic detergent and a metal chelating agent. The methods of the invention release large fragments of undegraded nucleic acids without physically disrupting the entire cell wall. The nucleic acids are released into solution without bursting the cells and are suitable for research and testing without further purification. The extraction method described herein is rapid, easy to perform, and applicable to a wide variety of cells, including microorganisms. Clinical samples may be screened for the presence or absence of a microorganism by heating the sample at 80-95 degrees Celsius in the presence of a non-ionic detergent and a metal chelating agent, adding to the sample a nucleic acid probe specific to the selected microorganism, incubating the sample under conditions which allow the probe to hybridize to released nucleic acid and detecting whether any hybridized probe is present. A kit for performing the nucleic acid extraction methods disclosed herein is also described.
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Claims
1. A method for extracting nucleic aids from a wide range of microorganisms, consisting essentially of the steps:
- a. contacting a sample containing one or more microorganisms in an aqueous suspension with a permeabilization reagent consisting essentially of a non-ionic detergent, from 0% to about 2% (w/v) of an anionic detergent, and a metal chelating agent; and
- b. heating the sample and permeabilization reagent together at about 80.degree.-100.degree. C. until nucleic acids are released from said microorganisms
2. A method as in claim 1 wherein the extracted nucleic acids further comprise a member of the group consisting of:
- a. nucleic acids having a nucleotide sequence consisting essentially of ribosomal RNA nucleotide sequences,
- b. nucleic acids having DNA nucleotide sequences from which said RNA nucleotide sequences are transcribed, and
- c. nucleic acids having nucleotide sequences complementary to said DNA nucleotide sequences.
3. A method as in claim 1 wherein said one or more microorganisms are each selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi.
4. A method as in claim 1, wherein said one or more microorganisms are each selected from the group consisting of Acintobacter species, Actinomyces species, Aerococcus species, Aeromonas species, Alclaigenes species, Bacillus species, Bacteriodes species, Bordetella species, Branhamella species, Brevibacterium species, Campylobacter species, Candida species, Capnocytophagia species, Chromobacterium species, Clostridium species, Corynebacterium species, Cryptococcus species, Deinococcus species, Enterococcus species, Erysielothrix species, Escherichia species, Flavobacterium species, Gemella species, Haemophilus species, Klebsiella species, Lactobacillus species, Lactococcus species, Legionella species, Leuconostoc species, Listeria species, Micrococcus species, Mycobacterium species, Neisseria species, Nocardia species, Oerskovia species, Paracoccus species, Pediococcus species, Peptostreptococcus species, Propionibacterium species, Proteus species, Psuedomonas species, Rahnella species, Rhodococcus species, Rhodospirillium species, Staphlococcus species, Streptomyces species, Streptococcus species, Vibrio species, and Yersinia species.
5. A method as in claim 1 wherein the non-ionic detergent is selected from the group consisting of polyoxyethylene alcohols and octylphenol-ethylene oxide condensates.
6. A method as in claim 5 wherein the concentration of the non-ionic detergent is between about 0.01% and 1%.
7. A method as in claim 5 wherein the concentration of the non-ionic detergent is about 0.07%.
8. A method as in claim 1 wherein the metal chelating agent is EDTA.
9. A method as in claim 8 wherein the concentration of EDTA is about 10 millimolar.
10. A method as in claim 1 wherein the pH of the permeabilization reagent is less than about 8.0.
11. A method as in claim 1 wherein the heating is carried out for about 1-30 minutes.
12. A method as in claim 9 wherein the heating is carried out for about 5 minutes.
13. A method as in claim 1 wherein the nucleic acids suitable for hybridization are RNA.
14. A method as in claim 1 wherein the nucleic acids suitable for hybridization are DNA.
15. A method as in claim 1 where the anionic detergent is selected from the group consisting of lithium lauryl sulphate and sodium dodecyl sulphate.
16. A method as in claim 15 wherein the concentration of the anionic detergent is about 0.05%-2%.
17. A method as in claim 16 wherein the concentration of the anionic detergent is about 1%.
18. A method as in claim 1 wherein the metal chelating agent is EDTA.
19. A method as in claim 1 wherein the sample and permeabilization reagent are heated for about 1-30 minutes.
20. A method as in claim 1 wherein;
- a. the non-ionic detergent is polyoxyethylene p-t-octylphenol at a concentration between about 0.01% and 1%;
- b. the anionic detergent is lithium lauryl sulphate at a concentration between about 0.05% and 2%; and
- c. the metal chelating agent is EDTA at a concentration between about 5 and 50 millimolar.
| 4486539 | December 4, 1984 | Ranki et al. |
| 4830969 | May 16, 1989 | Holmes |
| 5212059 | May 18, 1993 | Schwartz et al. |
| 5231015 | July 27, 1993 | Cummins et al. |
| 149514 | January 1985 | EPX |
| 547789 | November 1992 | EPX |
| 4349892 | May 1991 | JPX |
| WO92/ 07096 | October 1993 | WOX |
- Doi, et al., "Isolation of Nuclei from a Tetraploid Strain of Saccharomyces cerevisiae", J. Biochem., 75(5):1017-1026 (1974). Mifflin, Theodore E., "Use and Applications of Nucleic Acid Probes in the Clinical Laboratory", Clin. Chem., 35(9):1819-1825 (1989). Darnell, et al. RNA synthesis and processing in eukaryotes. Molecular Cell Biology, Scientific American Books, New York. pp. 322-323 (1986). Lindblom, et al. Rapid DNA purification for restriction fragment length polymorphism analysis. Gene Anal. Techn. 5:97-101 (1988). Ortlepp, et al. An improved boiling method for the preparation of bacterial plasmid and phage DNA. Gen Anal. Techn. 6:93-96 (1989). Stratagene Catalog. Gene characterization kits. p. 39 (1988). Derwent Abstract of Japanese Patent No. 4,349,892 to Toshiba KK. Meijer, et al. Recovery and identification of DNA sequences harboured in preserved ancient human bones. Biomedical and Biophysical Research Communications. 183(2):367-374 (1992). New England Biolabs Catalog, (1992), p. 73. Arnold, et al. Assay formats involving acridinium-ester-labeled DNA probes. Clin. Chem. 35(8):1588-1594 (1989). Sritharan and Barker, Jr. A simple method for dignosing M. tuberculosis infection in clinical samples using PCR. Molecular and Cellular Probes 5:385-395 (1991). Ulenhopp, et al. Viscoelastic Characterization of Single-Stranded DNA. Biophys. J. 15: 223-232 (1075) pp. 224-225. Godson, et al. Lysis of E. coli with a neutral detergent. Biochim. Biophys. Acta 149:476-488 (1967). Ulenhopp, et al., "Viscoelastic Characterization of Single-Stranded DNA", Biophys. J. 15:223-232,1975 pp. 224-225. Godson,et al., "Lysis of E. coli with a neutral deteregent", Biochim. Biophys. Acta 149:476-488 1967.
Type: Grant
Filed: Oct 18, 1995
Date of Patent: Jul 28, 1998
Assignee: Gen-Probe Incorporated (San Diego, CA)
Inventors: Kathleen A. Clark (Cardiff by the Sea, CA), Daniel L. Kacian (San Diego, CA)
Primary Examiner: Bradley L. Sisson
Attorney: Carlos A. Fisher
Application Number: 8/544,560
International Classification: C12N 108; C12N 1500; C12Q 168; C07H 2104;
